Reaction mass of cobalt olivine and crystalline silicon dioxide

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-11-06 to 2019-12-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

2019-06-18

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation Protocol: EpiOcular™ Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; for use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model

Version / remarks:

2015-06-29

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2019-10-23

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of cobalt olivine and crystalline silicon dioxide

EC Number:

701-439-7

Molecular formula:

Co2SiO4∙xSiO2 (0,7≤x≤1,5)

IUPAC Name:

Reaction mass of cobalt olivine and crystalline silicon dioxide

Test material form:

solid: particulate/powder

Details on test material:

- Test item identification: Olivine, cobalt silicate blue (Pigment blue 73)
- new chemical name: Reaction mass of cobalt olivine and crystalline silicon dioxide
- Substance type: inorganic pigment
- Storage conditions: Keep dry and container tightly closed. Keep container in an adequately ventilated storage

Test animals / tissue source

Details on test animals or tissues and environmental conditions:

JUSTIFICATION OF THE TEST METHODS AND CONSIDERATIONS REGARDING APPLICABILITY:
This in vitro method is recommended to identify chemicals that do not require classification for eye irritation or serious eye damage according to UN GHS (UN GHS “No Category”) without further testing within a tiered testing strategy from those requiring classification and labelling (UN GHS categories 1 and 2). Therefore, it can be used for regulatory purposes as an initial step in the bottom-up approach or as one of the last steps in a top-down approach to test eye irritation/corrosion potential. It is not intended to differentiate between UN GHS “Category 1” (serious eye damage) and UN GHS “Category 2” (eye irritation) which would require additional testing. Ocular irritation potential is predicted by the relative viability of the tissue after a single exposure to the test substance. Relative viability is determined by measuring the MTT dye to formazan conversion by the EpiOcular™ tissue construct after topical exposure to the test substance.

RhCE TISSUE CONSTRUCT USED: EpiOcular™ (Lot No.: 30639; Standard Assay Kit and MTT-100 kit; source: MatTek Corporation (82105 Bratislava, Slovakia))
The test was carried out with the EpiOcular™ reconstructed human cornea-line epithelium (RhCE) model (MatTek). The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified, highly differentiated squamous epithelium morphologically similar to that found in a human cornea. The EpiOcular™ RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinized surface, showing a cornea-like structure analogous to that found in vivo.

Please also refer to the field "Attached background material " below.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 ± 2 mg were applied to the tissue surface. The test item was tested neat.

Duration of treatment / exposure:

6 hours

Observation period (in vivo):

not applicable

Duration of post- treatment incubation (in vitro):

about 18 hours

Number of animals or in vitro replicates:

Number of EpiOcular tissues:
Test item: duplicates
Negative control: duplicates
Positive control: duplicates

Details on study design:

PRE-TEST FOR MTT INTERFERENCE
- To test if a test item directly reduces MTT, 1 ml of a MTT solution (1 mg/mL) including 50 ± 2 mg of the test item was incubated for 180 min under (37 ± 1.5°C, 5 ± 0.5% CO2).
- 50 µL deionised water in MTT solution was used as negative control.
- After incubation the change of colour was determined by the unaided eye.

PRE-TEST FOR COLOUR INTERFERENCE
- To check the colouring potential of the test item, 50± 2 mg of the test item was added to 1 mL of deionised water and mixed. 1 mL of deionised water was used as control (blank). Both were incubated for 60 min (37 ± 1.5°C, 5 ± 0.5% CO2).
- In parallel, 50 ± 2mg of the test item was added to 2 mL of isopropanol and mixed. A control (2 mL of isopropanol, blank) was run concurrently. Both were incubated for 3 hours at room temperature.
- After incubation the presence of the staining was evaluated by OD measurement.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE AND COLOUR INTERFERENCE
Since the OD of the test item in deionised water at 570 nm after blank correction was > 0.08 in the colour interference test and the test item interfered with MTT in the MTT interference test, the main experiment had to be performed with additional viable (CC), freeze killed (KC) and an additional third set of controls in order to avoid double correction. These additional tissues were designated Non-Specific Killed Control (NSKC) and run in duplicates. They are freeze-killed tissues which were treated with the test item but incubated in medium without MTT solution in the MTT assay.
- Negative control/ Deionised water treated tissues (NC_CC) without MTT solution in the MTT assay
- Test item treated tissues (TI_CC) without MTT solution in the MTT assay
- Negative control/ Deionised water treated freeze-killed tissues (NC_KC)
- Test item treated freeze-killed tissues (TI_KC)
- Test item treated freeze-killed tissues (TI_NSKC) without MTT solution in the MTT assay
At the end, the following data correction procedure for Viability plus Killed Control (KK), Colorant Control (CC) and Non-Specific Killed Control Tests (NSKC) was performed:
Corrected viability (%) = TI viability – TI_CC viability – (TI_KC viability – NC_KC viability) + TI_NSKC viability

DETAILS OF THE TEST PROCEDURE USED
Pre-warming:
- EpiOcularTM tissues were equilibrated at room temperature for 15 min. The inserts with the tissues were transferred into 6-well-plates containing 1.0 ml assay medium and incubated for 60 minutes (37 ± 1.5°C, 5 ± 0.5% CO2). Afterwards, the medium was changed and a further pre-incubation for approx. 17 hours (37 ± 1.5°C, 5 ± 0.5% CO2) follows.
- Treatment:
After pre-warming and prior to application of the test item respectively the controls, all tissues were pre-wetted with 20 µL Ca2+Mg2+free-DPBS and incubated for 30 min (37 ± 1.5°C, 5 ± 0.5% CO2).
Concurrent negative and positive control were applied at a volume of 50 µL and for the test item 50 ± 2 mg to the tissue surface and incubated for 6 h in assay medium (37 ± 1.5°C, 5 ± 0.5% CO2)
Afterwards all tissues were rinsed 3 times in 100 mL PBS and incubated for 25 min in 5 ml assay medium at room temperature in a 12-well plate (post-exposure immersion). At the end of this incubation the tissues were transferred into a 6-well plate with 1 ml assay medium and were incubated for a post exposure incubation for about 18 h (37 ± 1.5°C, 5 ± 0.5% CO2).
- MTT Assay:
Each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into the 24-well plate containing 300 µL of MTT solution and were incubated for 180 min (37 ± 1.5°C, 5 ± 0.5% CO2).
The inserts were removed from the 24-well plates after 180 minutes. The bottom of each insert was blotted on absorbent material, and then transferred into a pre-labelled 24-well plate containing 2 mL isopropanol so that no isopropanol was flowing into the insert on the tissue surface. For this,tissues treated with the test item were extracted from the bottom of the tissue only, to minimise any potential contamination of the isopropanol extraction solution with any test item that may have remained on the tissue. The concurrently tested negative and positive control substances were treated similarly to the tested item. Additional viable and NSKC tissues were incubated in 300 µl medium under standard conditions.
Each tissue was immediately extracted with isopropanol within 2-4 h while shaking at room temperature in the first experiment. In the second experiment each tissue was stored overnight at 2-8°C without shaking. After the extraction, the plates were placed on an orbital plate shaker and shaken for approx. 2.5 hours at room temperature to extract the MTT. At the end of the extraction period, the tissues were not pierced.
Then, the extracts were mixed and two 200 µL aliquots were transferred to a 96-well plate for OD measurement. 200 µL of isopropanol were added to the wells designated as blanks for 96-well plate.
-Measurement:
The optical density (OD570nm) was determined spectrophotometrically in duplicates by a microplate reader (Versamax® Molecular Devices). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

TEST ACCEPTANCE CRITERIA
The test meets acceptance criteria:
- mean absolute OD570 nm of the negative control is > 0.8 and < 2.5
- mean relative tissue viability of the positive control is < 50%
- relative tissue viability difference of replicate tissues is < 20%
- OD control values are not be below historically established boundaries/ positive and negative control mean values and acceptance ranges based on historical data.

DEMOSTRATING OF PROFICIENCY IN PERFORMING THE TEST METHOD BEFORE ROUTINE USE BY TESTING OF THE PROFICIENCY CHEMICALS
Prior to routine use of EpiOcularTM EIT for regulatory purposes, the laboratory demonstrated technical proficiency by correctly predicting the eye irritation potential of fifteen proficiency chemicals listed in Table 1 of OECD TG 492. The respective proficiency certificate given by MatTek is attached in the field "Attached background material" below.

DEMOSTRATING OF PROFICIENCY IN PERFORMING THE TEST METHOD BEFORE ROUTINE USE BY TESTING OF THE PROFICIENCY CHEMICALS
Prior to routine use of EpiOcularTM EIT for regulatory purposes, the laboratory demonstrated technical proficiency by correctly predicting the eye irritation potential of fifteen proficiency chemicals listed in Table 1 of OECD TG 492. The respective proficiency certificate given by MatTek is attached in the field "Attached background material" below.

DESCRIPTION OF DATA EVALUATION
1) The mean OD value of the two wells for each tissue and the blank control (ODBlk) was calculated (Mean [OD570] (well 1 and well 2).
2) The mean ODBlk was subtracted from each mean OD value of the two wells.
(Mean [OD570] blank corr. (well 1 and well 2)). These values were used for all further calculations below.
3) The mean OD of the two relating tissues for each test group (negative control (NC), positive control (PC)) and the test item (TI) was calculated with the blank corrected mean OD (Mean [OD570] of T1 and T2)
4) The percent viability of each test group relative to the negative control (= 100%) was calculated:
Viability (%) =100 × (mean OD_TI/PC/NC) / mean OD_NC)
5) The relative OD of each tissue per test group was calculated. 100 divided by the mean ODNC T1 and T2 x mean OD of each test group.
6) The difference of the viability values between duplicate tissues was calculated: The relative OD of T2 was subtracted from T1.

PREDICTION MODEL
If the test item-treated tissue viability is > 60% after exposure and post-exposure incubation relative to the negative control treated tissue viability, the test item is identified as not requiring classification and labelling according to UN GHS (No Category).
If the test item-treated tissue viability is ≤ 60% after exposure and post-exposure incubation relative to negative control treated tissue viability, no prediction can be made for this test item.

Results and discussion

In vitro

Results

Irritation parameter:

other: % tissue viability (mean)

Value:

89.07

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

TEST FOR MTT INTERFERENCE
Optical evaluation of the MTT-reducing capacity of the test item with MTT-reagent showed purple colour. Therefore, an additional test with freeze-killed tissues was necessary in the min experiment.

TEST FOR COLOUR INTERFERENCE
In the colour interference the test item changed colour when mixed with deionised water. The mean OD of the test item in deionised water was > 0.08 and therefore, an additional test with viable tissues without MTT addition was necessary in the main experiment.

ACCEPTANCE OF RESULTS:
- mean absolute OD570 nm of the negative control is > 0.8 and < 2.8 (2.289)
- mean relative tissue viability of the positive control is < 50% (32.60%)
- relative tissue viability difference of replicate tissues is < 20% (0.05 p.p. to 12.85 p.p.)
- The upper limit of the historical control range OD of the negative control were slightly exceeded, but since the OD was within the recommended OECD 492 range (> 0.8 and < 2.8), this minor deviation was judged as irrelevant and does not compromised the validity of this study.
- The OD control values of the positive control were within the historically established boundaries.
- positive and negative control mean values and acceptance ranges based on historical data.
The acceptance criteria were met.

Please also refer to the field "Another information on results incl. tables" below.

Any other information on results incl. tables

Results after treatment with Cobalt silicate olivine & silica polymorphsand the controls for 6 hours:

Test Group	Tissue No.	Well 1 [OD570]	Well 2 [OD570]	Mean [OD570] (Well 1 and well 2)	Mean [OD570] blank corr. (Well 1 and well 2)	Mean [OD570] of T1 and T2	Tissue viabil. [%]	Viabil. of T1 and T2 [%]	Diff. of viabil. between T1 and T2 [p.p.]	Mean viability of test item after data correct. procedure [%]
Blank		0.037	0.037	0.037						89.07*
Negative Control	1	2.379	2.331	2.355	2.318	2.289	100.0	101.3	2.61
	2	2.283	2.308	2.296	2.259			98.7
Positive Control	1	0.745	0.727	0.736	0.699	0.746	32.60	30.6	4.10
	2	0.828	0.832	0.830	0.793			34.7
Test Item	1	2.230	2.206	2.218	2.181	2.034	88.88	95.3	12.85
	2	1.943	1.905	1.924	1.887			82.5
Negative Control Viable Tissues (NC_CC)	1	0.043	0.042	0.042	0.005	0.004	0.19	0.2	0.07
	2	0.041	0.041	0.041	0.004			0.2
Test Item Viable Tissues (TI_CC)	1	0.042	0.042	0.042	0.005	0.004	0.19	0.2	0.05
	2	0.041	0.041	0.041	0.004			0.2
Negative Control Freeze killed Tissues NC_(KC)	1	0.086	0.084	0.085	0.048	0.049	2.14	2.1	0.12
	2	0.087	0.088	0.087	0.050			2.2
Test Item Freeze killed Tissues (TI_KC)	1	0.082	0.080	0.081	0.044	0.045	1.98	1.9	0.10
	2	0.084	0.084	0.084	0.047			2.0
Test Item NSKC (TI_NSKC)	1	0.041	0.042	0.042	0.004	0.005	0.23	0.2	0.06
	2	0.043	0.043	0.043	0.006			0.3

* Corrected mean viability= TI viability –TI_CC viability – (TI_KC viability – NC_KC viability) + TI_NSKC viability

 

Historical Control Data 

Positive Control (Methyl acetate)		Negative Control OD at 570 nm (Deionised water)
Mean Viability	27.15%	Mean Absorption	1.67
Standard Deviation	9.89 p.p.	Standard Deviation	0.28
Range of Viabilities	6.73%—42.54%	Range of Absorbance	1.02—2.09
Mean Absorption OD at 570 nm	0.45
Standard Deviation	0.17
Range of Absorbance	0.08—0.78
Data of 30 sets of controls shared between 69 studies performed from June 2016 until November 2019. (p.p.—percentage points)

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, Cobalt silicate olivine & silica polymorphs does not require classification and labelling for eye irritation or serious eye damage according UN GHS.